Combinations/ Antibody-Drug Conjugates

The need for multiple assay depends on the properties/characteristics of the mAb/conjugate and if other conjugates are manufactured at the same facility.

Charge based assays may not be possible/meaningful after conjugation lysine residues.

Antibody-Drug Conjugate: Purity

Largely the same methods as before conjugation.

SDS-PAGE/CGE, SEC-HPLC, charge based.

Charge based assays may not be possible/meaningful after conjugation to lysine residues.

Need to control for aggregates and fragments.

Need to understand what is stability indicating.

Antibody-Drug Conjugate: Potency

Mechanism of action is determined.

Needs antigen binding for unconjugated mAb.

Needs to demonstrate conjugation does not affect antigen binding.

If conjugation process is well controlled, antigen binding assay may be eliminated provided cytotoxicity assay is robust.

Clinical ADC manufacturing must be executed in an aseptic biological manufacturing environment which operates under cGMP since the antibody modification and cytotoxin conjugation reaction are usually executed under process conditions which will support growth of environmental microbial contaminates. Batch failures resulting from human error or equipment failure are typically low for biological manufacturing.

In the antibody modification suite the naked antibody raw material which is typically frozen at -20°C is thawed and the storage buffer is exchanged via tangential flow filtration. Buffer exchange is often required to alter the pH, salt concentration and remove excipients used to improve storage stability in the bulk antibody solution which may hinder or alter the performance of the conjugation reaction. These steps include the isolator used for weighing and preparation of the solution containing the cytotoxin (Weighing and Dispensing Containment Isolator, Containment Barrier Isolator, and Aseptic Containment Isolator) and the conjugation suite, in which the cytotoxin is combined stoichiometrically with the antibody linker complex. Bulk ultrafiltration/difiltration (UF/DF) or chromatography is then employed to remove process contaminants such as free cytotoxin and organic solvent.

In the isolator suite cytotoxins can be weighed and solubilized in organic solvent for introduction into the antibody conjugation suite via a hard piped transfer line into the conjugation vessel. The isolator permits safe handling of the cytotoxins in a negative pressure environment and a means of addressing the electrostatic properties of cytotoxins through humidity control. Since the interior of the hood comes in direct contact with potent cytotoxins the hood is designed with a clean in place (CIP) system that permits inactivation of the cytotoxin and/ or multiple detergent and rinse cycles to remove cytotoxin contamination.

In the conjugation suite the coupling between antibody and the cytotoxin is performed in a sealed temperature and pH controlled stainless steel tank with nitrogen over-lay. Special equipment modification in this area such as double mechanical seals, overflow trays and positive displacement pumps with both primary mechanical seals and secondary water seals strengthen the primary containment envelope to protect employees working in the suite. After removal of the process contaminants the buffer containing the ADC is transferred to the formulation suite for transition into the BDS formulation buffer.

All liquid and solid waste from the process are then inactivated and incinerated on site depending on process requirements. Following API concentration adjustment and excipient addition in the formulation suite the BDS is then stored frozen at -20 to -60?C using a controlled rate shell freezer or aseptically dispensed into multiple PETG bottles for bulk storage.

Manufacturing of ADCs

Esco Pharma has a wide array of manufacturing products which can be used specifically for the development of the next generation of pharmaceuticals.

The general workflow for the production of ADCs:

Sample Preparation

Pharmacon™ Downflow Booths

For the handling of raw materials, Esco Pharma offers the Downflow Booth, which is primarily used in the pharmaceutical industry to provide a safe working area for operators to protect them from hazardous, sensitizing, or toxic substances during manual handling of powders and solvents.

Formulation

Weighing & Dispensing Containment Isolators (WDCI)

Weighing and Dispensing Isolators are advanced containment systems providing controlled negative pressure environments to maximize personnel protection. This equipment is especially designed for the weighing and dispensing of APIs, and as for ADC production, the isolator can be used for the mixing and blending with excipients. BioVap™ bio-decontamination system can also be installed in the equipment, wherein atomized hydrogen peroxide is sprayed throughout the equipment, thus sterilizing the system.

Formulation isolators are offered by Esco Pharma to fit the needs of the client for ADC manufacturing. Auto-loading and unloading systems can also be integrated in isolators for a better and faster workflow. Utilization of half suits for manual loading and unloading can also be installed, as well as the addition a blender, mixer, centrifuge, sieve and a lyophilizer for the production of the active pharmaceutical ingredient (API) and its salts. The integration of freeze dryers is also applicable, for the finalization and storage of the ADCs.

Esco TideCell® Bioreactor System

Esco offers tide motion bioreactors for the production of monoclonal antibodies (mAbs), wherein it features the same bioprocess method from seed preparation to production scale. The Esco TideCell® Bioreactor System is the world's largest linearly scalable single-use bioreactor, from seed preparation to 5000 liters with closed automated cell harvesting.

Filling Process

Antibody drug conjugates are produced in either a small or a large scale, after being processed in different stages. Esco can provide these needed qualifications and has also established collaborations with companies which provide filling lines, fit for potent formulation and is also ideal for a small to a medium batch production.

Active Open Restricted Access Barriers System (oRABS)

Transfer of products to the main process chamber happens via a Rapid Transfer Port (RTP). The entry and exit of vials happen inside an ISO Class 6 environment, but the whole process happens inside an ISO Class 5 production. The Active Open Restricted Access Barriers System (oRABS) is an example of a containment system which can serve as a fully integrated system for the filling of injectable biopharmaceutical products into ready-to-use containers, including vials, syringes, and cartridges. It can serve different cleanliness classifications as required, depending on the state of the product to be processed inside.

Sterility Testing

General Processing Platform Isolator (GPPI)

Esco General Processing Platform Isolator (GPPI) which is a highly adaptable, unidirectional laminar airflow isolator that can be used for sterility testing or other processes that require an ISO Class 5 (Grade A) aseptic environment. The GPPI's advanced control system allows the operator to select either positive or negative chamber pressure as well as single pass or recirculating airflow patterns.

Esco Pharma provides aseptic and containment isolators, giving the best personnel, product, and environmental protection. Customization of equipment to fulfil the client's needs is what Esco can truly offer, so that provision of a truly integrated solution will be available whenever needed. With continuous innovation, quality is built into the products with the use of Esco's equipment.

Raw Material Supply Chain

ADC(s) have the most extensive supply chain of any biological requiring sourcing of an antibody, linker and potent small molecule cytotoxin. Most biopharmaceutical companied currently developing ADC(s) prefer to use an intact monocolonal antibody targeting agent. The cell bank used to produce the antibody will require extensive virus testing to prove the cell bank is free from adventitious agents. Also the associated downstream purification process for anybody will require evidence that the manufacturing process can inactivate, clear and remove virus if contamination is present. The potent cytotoxins currently used in ADC manufacturing could be semi-synthetic (fermentation derived intermediate) or fully synthetic (chemical synthesis).

Culture Production Factors

Cell lines

The great majority of commercial mAbs are produced in Chinese hamster ovary (CHO) and NS0 cells, originating from plasmacytoma cells that were modified until IgG generation in non-secreting B cells.

Microorganisms modified by genetic engineering techniques have attracted much focus since these cells are simpler to handle and easier to modify compared to animal cells. Other advantages of production methods using genetically modified organisms are that these cell have well-defined expression systems, and the production methodology is reproducible and easy to validate. Modified yeast cells, such as Pichia pastoris have a great potential for usage since these cells are known to achieve high secretion levels of heterologous proteins.

Culture Medium

Cultivation media for mammalian cells must have a complex content of ingredients ranging from amino acids to trace elements. To supply the cellular demand of these nutrients, the culture medium uses serum.

Super Plus™ is a cell culture supplement designed to reduce or eliminate the requirement for serum in the basal medium of cell culture. Super Plus™ also minimizes culture variance due to inconsistent quality between serum batches. Super Plus™-added medium supports growth of many cell lines, including MDCK, VERO, BHK, ST, PK-15, HEK-293, and CHO.

Growing conditions can directly influence the cell growth and production levels of molecules of interest. Usually, mammalian cell culture conditions for mAb production are very well defined: 37° C, pH 7.15, and dissolved O2 (OD) levels at 30-60%.

Production Platforms

Batch Production

The simplest of all the production platform is the batch production, which consists of a closed system were a bioreactor is sterilized and prepared with a medium containing all the nutrients needed for cellular growth and product manufacturing.

Continuous Fermentation

There are two types of continuous production: chemostat cultures and perfusion cultures. For chemostat cultures, fresh medium is added to the bioreactor and fermented medium is removed along with cells at a constant flow rate so that the culture volume remains unchanged. The flow rate controls cellular growth and when these two variables are equal, the bioreactor reaches equilibrium-cell concentration, nutrient concentration, and product concentration are held constant. To avoid viable cell loss along with constant outflow of the by-products of cell metabolism, many manufacturing plants have developed a cell-recycling system and thus, the perfusion method was developed where cells are kept inside the bioreactor.

Fed-batch processes

This is by far the most utilized at industrial scale. In this process, the cell density reaches 8-12 x 10 6 cells/mL, and cell viability in the bioreactor is enhanced by controlled nutrient addition at specified intervals.

Production Systems

Production Systems for cells in suspension cultures

Stainless steel stirred tank bioreactors are the most consolidated type of bioreactor used for industrial mAb production and consist of baffle-stirred tanks linked to rotor systems. It is a consolidated system, and there is a lot of knowledge and experience surrounding this technology, acquired by its vast industrial use beyond production using mammalian cells.

StirCradle is a desk-type bioreactor/fermenter for microorganism culture. By combining the vessel, workbench, controller and peristaltic pumps into one integrated system, this patented system effectively provides the most capacity for a small footprint. The StirCradle is ideal for research and development involving microorganism or animal and plant cell cultures.

The StirCradle-Pro is a fully stainless steel fermenter/bioreactor system designed with an automated 5-step cycle SIP system for both culture medium and reactor vessel. VacciXcell offers a wide range of standard reactor capacities from 20L up to 1000L; however, customized reactor sizes are available should the users require.

Air lift reactors are also broadly used for the industrial production of mAbs. The reactor consists of tanks with a bubble column inside, and air is injected into the column base. The air flows through the column’s length to the top of the bioreactor as degassed culture medium flows in the opposite direction to the reactor bottom. This creates a constant gentle mixing of the medium as well as proper culture aeration, annulling part of the shear stress caused by other stirring systems.

Disposable bioreactors bought many changes for mAb manufacturing. At the end of the process, the bioreactor is discarded and replaced by a new clean and sterile one. This eradicated cross contamination between batches and decreases the time consumed with the equipment preparation between batches.

CelCradle™ is a disposable bioreactor capable of high-density cell culture for protein expression, virus, and monoclonal antibody production. It is designed based on the concept of bellow-induced intermittent flow of media and air through porous matrices, where cells reside. This provides a low shear, high aeration, and foam-free culture environment.